Basic introduction of Real-time PCR and
LightCycler® 480 system

Jing-yuan Chen

Graduate Institute of Veterinary Pathobiology

08 16, 2019

Online slide

Polymerase chain reaction
(PCR)

PCR

  • Thermal cycle
PCR

Source: Polymerase chain reaction

Amplification curve

Four pahses for PCR

  • Ground
  • Expoential
  • Linear
  • Plateau


  • 2n / cycle

PCR stage

Reference DOI: 10.1100/tsw.2011.124.

Crucial factors for PCR efficiency

  • Quality of DNA template
  • DNA polymerase reality
  • dNTP concentration
  • Primers design, concentration…
  • Many other factors!

    • Theoretically!

Real-time PCR

Real-time PCR machines

Roche Bio-Rad Applied Biosystems Qiagen
LightCycler®480
LC480
CFX96 TouchTM
CFXConnect96
StepOneTM
ABIStepOne
Rotor-Gene Q
Qiagen Roter gene q

Basic introduction

Applications

Gene detection Gene expression
  • Species identification
  • Pathogens screening
  • Diseases diagnosis
  • Antibiotic resistance gene
  • Genotyping
  • Food safety analysis
  • Gene knockdown
  • Pharmacy researches

Two major detection format

  • Non-specific dsDNA binding dyes

  • Sequence-specific fluorescence detection chemistry

SYBR green I

SYBR green 1

Indiscriminate binding into any dsDNA
  • Primer dimers
  • Sample DNA quality
  • Melting curve analysis (Tm calling analysis)

Reference DOI: 10.1038/sj.leu.2402922

Melting curve analysis

Melting analysis

Reference DOI: 10.1002/9780470027318.a1406.pub2

Hybridization probe

  • Fluorescence Resonance Energy Transfer (FRET)

FRET

  • TaqMan probe

TaqManProbe

Reference DOI: 10.1038/sj.leu.2402922

all format

Reference DOI: 10.1007/s13197-016-2205-0

How to design my real-time PCR?

Resource

qPCR design

Free websites

Primer3 線上 primer 設計
http://bioinfo.ut.ee/primer3-0.4.0/
Beacon 線上 primer 設計
http://www.premierbiosoft.com/qOligo/Oligo.jsp?PID=1
mfold 檢查目標產物結構
http://unafold.rna.albany.edu/?q=mfold/download-mfold
clustal omega 多重序列比對
https://www.ebi.ac.uk/Tools/msa/clustalo/
Reference DOI: 10.1002/pro.3290

Example (I)

曾經有過這樣的設計 (142-232),看起來好像不錯!?

CSFV qPCR

Exapmle (II)

結果,因為立體結構的關係,使得擴增效率非常差!

  • 設計實驗以後,利用免費資源看一下可能的狀況!

Structure1Structure1Structure1Structure1

Data analysis

flowchart

Reference DOI: 10.1016/j.bdq.2014.08.002

Quantification

  • Cp calculation

  • Abosolute

  • Relative

  • Tm calling analysis (Melting curve analysis)

Cp calculation

  • Crossing poing (Cp) = Cycle quantification (Cq) = Cycle threshold (Ct) = Take-off point (TOP)
  • 各家儀器設備設定值不同而有所差異!!
  • MIQE

qPCR_Ct

Picture source:What Is A Cycle Threshold (Ct) Value In qPCR?

PCR efficiency

PCR efficiency

Reference: http://www.gene-quantification.de/pfaffl-qPCR-efficiency1.gif

PCR efficiency (I)

  • PCR product number at the number of Cp

\(N = N_{0}\times E^{Cp}\)

  • Linearised

\(LogN = LogN_{0}+Cp\times LogE\)

  • Calculate the “Cp” and describe the PCR amplification

\(Cp=-(\frac{1}{LogE})LogN_{0}+\frac{LogN}{LogE}\)

  • Slope of this equation!
  • Draw the plot…

PCR efficiency (II)

  • PCR efficiecy “E” can be described as
  • \(E = 10^{-\frac{1}{Slope}}\)
  • \(E(\%) = -1 + 10^{-\frac{1}{Slope}}\)

Absolute quantification

Second-derivative maximum method
Standard method
2nD
Fit point method
Optional, user define
fitpoint

Reference DOI: 10.1002/9780470027318.a1406.pub2

Relative quantification (I)

  • Determination of mRNA expression level
  • Gene dosage quantification
  • Relative to reference gene and/or calibrator sample
Relarive Protein expression
  • SA: stalbe angina
  • ACS: acute coronary syndrome

Reference DOI: 10.5114/aoms.2014.44871

Relative quantification (II)


\(Relative\ ratio = \frac{Target\ gene\ concentration}{Reference\ gene\ concentration}\)

Reference gene:

  • Unaffected by experiment conditions (non-regulated)
  • Wide variety of cellular processes
  • One or more reference can be used

Reference gene selection

Reference DOI: 10.1016/j.bbrc.2003.11.177 (被引用1,367次)

Relative quantification (III)

Optional:

\(Calibrator\ Normalize\ Ratio = \frac{\frac{Target\ gene\ concentration}{Reference\ gene\ concentration}(Sample)}{{\frac{Target\ gene\ concentration}{Reference\ gene\ concentration}(Calibrator)}}\)

Calibrator:

  • Correct the differences in detection sensitivity between Target / Reference gene and several experiments (e.g., long-term studies)

  • Not treated cell line

  • Sample before experiment

Relative quantification (IV)

  1. Efficacy = 2 → (\(2^{\Delta\Delta CT} method\))
  2. Efficacy adjust (linear) - using standard curve

Relative quantification (V)

Relative quantification

Reference: Roche 教育訓練專員講義

Tm Calling analysis

Tm Calling

Analysis tools

  • LightCycler 480 Software

LC480 software

Resource fo analysis tools

Analysis toolsAnalysis platforms

The MIQE guidelines
for Real-time PCR

Different System

Different results?

Different Melting cruve

Reference DOI: 10.1016/j.mcp.2010.04.002

MIQE

  • Minimum Information for Publication of Quantitative Real-Time PCR Experiments, (2009)

  • MIQE & PCR (2016, Jun)

MIQE 2016

Ohter guides

Other guides

LightCycler® 480 Instrument

General of LC480

LC480 General

LC480 front
LC480_fans

Enviroment parameters

Enviroment parameters

  • 機器使用時,請將冷氣開啟,以維持溫度 (大約 26˚C 左右)。
  • 冷氣開啟時,請注意隨手關門,電費不便宜!

LightCycler® 480 (I)

LC480 外觀

LC480 light

LightCycler® 480 (II)

plate loader 1

plate loader 2

Sample capicity

Whit plate

限定使用Roche原廠耗材!
(盤子與封模,唯一限定的材料)

LightCycler® 480 (III)

  • Thermal cycler

Thermal cycler

Thermal base

LightCycler® 480 (IV)

  • Optical system LC480 cartoon

LC480 filters

LightCycler® 480 - Filter set

Filter set

LightCycler® 480 - Xenon lamp (氙)

Xenon lamp

一定要遵守這些事

請注意!

  • 使用前登記,安排實驗次序
  • 注意空調,維持機器運作時的室內溫度
  • 限定使用原廠白盤及封膜! (未遵守者禁止使用)
  • 燈泡很貴!請算好操作的時間,不要開著機器讓他燒!

Thank you!